The present invention relates to cytofectins comprising complex amphiphilic lipids in which a bioactive agent which is recognized by biological systems is joined to a Rosenthal Inhibitor core structure through an alkyl linking group. A number of alkyl linking groups are disclosed, including carboxy, carbamyl, ureyl, thioureyl, and guanidyl cytofectins.
A second aspect of the present invention relates to such cytofectins having a primary amine within 8 carbons of the quaternary nitrogen.
A third aspect of the present invention relates to such cytofectins wherein the biologically active moiety is an amino acid or peptide selected from those moieties which are non-polar, polar and uncharged, and negatively charged at physiological pH.
A fourth aspect of the present invention relates to such cytofectins wherein the biologically active moiety contains alternative amino acids which are not generally found in natural organisms.
A fifth aspect of the present invention relates to disubstituted RI cytofectins having two heterosubstituted groups on the quaternary nitrogen.
A sixth aspect of the present invention relates to ether cytofectins.
Cationic lipids are amphiphilic molecules having a lipophilic region, commonly comprising one or more hydrocarbon or alkyl groups, and a hydrophilic region comprising at least one positively charged polar head group. Cationic lipids are useful for facilitating the transport of macromolecules through the plasma membrane of cells and into the cytoplasm by forming net positively charged complexes. The process, which can be carried out in vivo as well as in vitro, is known as transfection, and the cationic lipids used in such techniques are known as cytofectins.
Cytofectins which enhance transfection efficiency as little as 3 fold over that observed with naked DNA are beneficial, although preferably transfection efficiency is increased 5-10 fold, and more preferably transfection efficiency is enhanced more than 10 fold.
Typically, cytofectins are combined with a neutral zwitterionic lipid such as a phospholipid, because it has been found that the two amphiphilic lipid species in combination are able to form vesicles comprising ordered lipid bilayers that are more effective at transfection than the cytofectin alone. These vesicles, or liposomes, have multiple positive charges on the surface which allow them to form a complex with a polynucleotide or other anionic molecule such as negatively charged proteins. Remaining net cationic charges on the surface of the polynucleotide/cytofectin/neutral lipid complex are capable of strong interaction with the predominately negative charge of the cell membrane surface.
Apart from the basic features of amphiphilic properties and the polar head group, cytofectins have considerable structural diversity in the lipophilic and hydrophilic regions. Many different cytofectin species have been synthesized for use in transfection and are now commercially available. Such cytofectins include, for example, Lipofectin(trademark), Lipofectin ACE(trademark), LipofectAMINE(trademark), Transfeactam(trademark), and DOTAP(trademark). The structural diversity of effective cytofectins reflects, in part, the observation that structure-function-recognition aspects of cytofectins differ with respect to distinct applications in cells. Experience with cytofectins structurally similar to the DOTMA compounds indicates that transfection activity depends in part on the cell type transfected (Felgner et al. J. Biol. Chem. 84:7413-7417, 1987; Wheeler et al. Biochem. Biophys. Acta, in press). Particularly, cationic lipids comprising spermine substitution of the ammonium groups proved more effective than DOTMA for transfection of some cell lines. This phenomenon suggests that effective transfection depends not only on passive fusion of the cationic lipid complex with the structural lipid bilayer of the plasma membrane, but on specific cellular characteristics and interaction between cell components and the individual cationic lipid species.
Structural variants among cytofectin species are therefore an indication of a more sophisticated understanding of the multiple and complex interactions of cytofectins with cells, and an effort on the part of investigators to take advantage of one or more of these interactions.
DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium, disclosed in U.S. Pat. No. 5,049,386 to Epstein, was one of the first cationic lipids developed, and lipids of this group have become reference compounds in evaluating comparative cytofectin potency in the development of new structural variants. DOTMA lipids are characterized by a propanaminium group having a quaternary nitrogen, which provides the cationic site of the molecule, together with a pair of C18 hydrocarbons that are ether-linked to the propyl backbone of the molecule. The quaternary nitrogen is trisubstituted with relatively shorter alkyl chains, such as methyl groups. A structurally similar cationic lipid, 1,2-bis(oleoyloxy)-3-3-(trimethylammonia)propane(DOTAP), comprises acyl, rather than ether-linked alkyl groups, and is believed to be more easily metabolized by target cells.
Some species of cationic lipids, for example, ammonium salts directly substituted by alkyl or acyl groups, were developed primarily for purposes of economy (U.S. Pat. No. 5,279,833 to Rose). Others were developed in an effort to provide less toxic effects; for example, a highly biocompatible cytofectin prepared from phosphatidylcholine and sphingomyelin: 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine(Avanti Polar Lipids, Inc. Alabaster, Ala., Cat. Nos. 890700-706).
U.S. Pat. No. 5,264,618 to Felgner et al., the contents of which are incorporated herein by reference, discloses cytofectins that are structurally similar to the Rosenthal Inhibitor (RI) of phospholipase A (Rosenthal et al., J. Biol. Chem. 235:2202-2206, 1960) and diacyl- or alkyl/acyl-species thereof. The RI based series of compounds are known by acronyms having the pattern: DORIE (C18); DPRIE (C16); and DMRIE (C14). These acronyms imply a common basic chemical structure; for example, DMRIE is 1-propanaminium, N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-bromide, (xc2x1)-(CAS registry:146659); the others differ in their substituent alkyl groups. These cytofectins, having a polar hydroxyethyl substituent on the quaternary ammonium group, provide more effective transfection in many cases than DOTMA type compounds. A study of the effect of varying substituents at the hydroxyalkyl moiety and variation of alkyl chain lengths on the transfection efficacy of the RI cytofectins is presented in Felgner et al. (J. Biol. Chem. 269:2550-2561, 1994). Again, the studies showed that the optimum hydroxyl alkyl chain length is cell-type dependent.
The conversion of DMRIE to xcex2AE-DMRIE (Wheeler et al., Biochem. Biophys. Acta, in press) has been found to have a significant effect on cytofectin activity. DMRIE, which has a quaternary nitrogen adjacent to a primary alcohol, thus imparting a pH independent positive charge, is one of the most active cytofectins now known. However, the substitution of a primary amine group for the alcohol on DMRIE to give xcex2AE-DMRIE was found to form DNA complexes that are structurally distinct from those with DMRIE, and xcex2AE-DMRIE is able to transfect many cell lines effectively in the absence of helper co-lipids. The observation that a single substitution in the cytofectin skeleton can provide marked changes in transfection properties suggests that other modifications can bring about similar improvements in gene delivery.
Continuing studies of the transfection event indicate that cationic lipids may facilitate not only entry of the functional molecule into the cytoplasm of a cell, but may also provide additional beneficial capabilities; for example, protecting the functional molecule from lysosomal degradation, facilitating entry into the nuclear compartment, or even preventing the degradation of the RNA transcription product by cytoplasmic enzymes. These functions of cationic molecules are believed to be related to specific structural features. Accordingly, there is a need for cytofectins that are particularly suited to transfection of foreign molecules into specific cell types. There is also a need to develop cytofectins that are able to perform specific intracellular functions.
One embodiment of the present invention is a compound of the formula 
wherein
R1 and R2 are independently H, linear or branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4.
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
R5 has the structure 
wherein
Z is selected from the group consisting of O, S, NR1, NH, Se, and CR7R8;
R6 is selected from the group consisting of absent, H, R1, R2, R3 and R4;
n is 1 to 6;
m is 1 to 10;
Y is a pharmaceutically acceptable anion; and
R7 and R8 independently or in combination are H or alkyl groups as defined for R1 and R2;
wherein if Z is O, n is 1, and m is 3, then R6 is selected from the group defined for R3 and R4 and wherein R1 and R2 are not both H.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are C10 to C20 alkyl or alkenyl groups, Z is O and R6 is an amino acid or peptide linked to Z as an ester.
In another aspect of this embodiment, Z is O, R1 and R2 are identical and are selected from the group consisting of C14H29 and (CH2)8CHxe2x95x90CH(CH2)7CH3, and R3 and R4 are methyl.
In a further aspect of this embodiment, R1 and R2 are saturated or unsaturated C10-C18 alkyl groups.
In another aspect of this embodiment, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In some of these compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In other compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a compound of the formula 
wherein
R1 and R2 are independently H, linear or branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
wherein R5 has the structure 
wherein
R6, or R6 together with R7, is selected from the group defined for R1, R2, R3 and R4 and optionally further comprises a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono, di- or polysaccharide, or other bioactive or pharmaceutical agent;
R8 is absent, or is H or an alkyl group selected from the group consisting of R1, R2, R3 and R4 and wherein R8 may be joined to R6 or R7 so as to form a ring;
W is O, NR10, NH, S, or Se;
R10 is an alkyl group as defined for R1 and R2;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion;
wherein R1 and R2 are not both H.
In some aspects of this embodiment, m is 2-10.
In some aspects of this embodiment, R1 and R2 are saturated or unsaturated C10-C18 alkyl groups. In some of these compounds, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In other compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In additional compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a compound of the formula 
wherein
R1 and R2 are independently H, linear or branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms is 0-4, n is 1-6, and m is 1-10; and
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
wherein R5 is xe2x80x94Oxe2x80x94R6 such that R5xe2x80x94Oxe2x80x94R6 comprises an ether linkage, wherein R6 is selected from the group consisting of amino acids, peptides, polypeptides, proteins, nucleic acids, nucleotides, polynucleotides, monosaccharides, disaccharides, polysaccharides, bioactive agents, pharmaceutical agents, and linear or branched, unsubstituted or substituted C1-23 heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion; and
wherein R1 and R2 are not both H.
In some aspects of this embodiment, m is 2-10.
In some aspects of this embodiment, R1 and R2 are both alkyl groups or are both alkenyl groups and R6 comprises an amino acid or peptide selected from the group consisting of amino acids and peptides which are non-polar, amino acids and peptides which are polar and uncharged, and amino acids and peptides which are negatively charged at physiological pH. In some of these compounds, R1 and R2 are both alkyl groups or are both alkenyl groups and R6 comprises a bioactive moiety.
In other compounds, R5 is selected from the group consisting of monosaccharides, disaccharides, and polysaccharides.
In further compounds, R1 and R2 are saturated or unsaturated C10-C18 alkyl groups. In some of these compounds, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In additional compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In further compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a compound having the structure 
wherein
R1 and R2 are independently H, linear or branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
wherein R5 is selected from the group defined for R3 and R4 and optionally further comprises a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, monosaccharide, disaccharide or polysaccharide, or other bioactive or pharmaceutical agent; and
Z is NH, or S;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion;
wherein R1 and R2 are not both H, and
wherein if Z is NH and n is 1 and m is 2 to 6, and R1 and R2 separately or together are C1-C23 alkyl or C(O)xe2x80x94C1-C23, and R3 and R4 separately or together are H or unbranched alkyl C1-C6, then R5 is not xe2x80x94(CH2)zNH2 where z is 2-6; or xe2x80x94(CH2)3xe2x80x94NHxe2x80x94(CH2)4xe2x80x94NH2; or xe2x80x94NHxe2x80x94(CH2)3xe2x80x94NHxe2x80x94(CH2)4xe2x80x94NH(CH2)3xe2x80x94NH2, C(O)-fluorescein, or 
where p is 2-5, Z is H or other groups attached by amide or alkyl amino groups.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are saturated or unsaturated C10-C18 alkyl groups. In some of these compounds, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In other compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In additional compounds, R3 and R4 are methyl groups. Another embodiment of the present invention is a compound having the structure: 
wherein
R1 and R2 are independently H, linear, branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene, or heteroalkyl groups having from 0 to 6 sites of unsaturation, or cyclic or aryl groups, said cyclic or aryl groups containing up to five heteroatoms, wherein the substituent groups are selected from the group consisting of xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein k is 0 to 4, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms;
R3 and R4 are independently linear, branched, unsubstituted or substituted C1-23 alkyl, acyl, alkene, or heteroalkyl groups having from 0 to 6 sites of unsaturation, or cyclic or aryl groups, said cyclic or aryl groups containing up to five heteroatoms, wherein the substituent groups are selected from the group consisting of xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein k is 0 to 4, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4;
R5 is absent, H or an alkyl group as defined for R1 and R2; R5 through R10 independently or in combination are absent, or are H or alkyl groups as defined for R1 and R2 and, optionally, further comprise a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono-, di- or polysaccharide, or other bioactive or pharmaceutical agent;
G is absent, O, N, S or Se;
Z is O, N, S, or Se;
A is O, N, S, Se, or C;
n is 1-6;
m is 1-10;
Y is a pharmaceutically acceptable anion;
wherein if G is N and Z is O, then A is not C;
wherein if G is O and Z is O then A is not C;
wherein if G is absent, Z is O, A is O, R6 and R7 are absent, n is 1, and m is 3, then R8 is not absent or H;
wherein R1 and R2 are not both H;
and wherein if G is NH and n is 1 and m is 2 to 6, and R1 and R2 separately or together are C1-C23 alkyl or alkenyl or C(O)xe2x80x94C1-C23 alkyl or alkenyl, and R3 and R4 separately or together are H or unbranched alkyl C1-C6, and Z is O then A is not fluorescein, or 
where
p is 2-5, Z is H or other groups attached by amide or alkyl amino groups.
In some aspects of this embodiment, m is 2-10.
One aspect of this embodiment is compounds having a primary amine within 8 atoms of the quaternary nitrogen.
In another aspect of this embodiment, if any of R5-R10 are amino acids or peptides they are selected from the group consisting of those amino acids and peptides which are non-polar, amino acids and peptides which are polar and uncharged, and amino acids and peptides which are negatively charged at physiological pH.
In yet another aspect of this embodiment, if any of R5-R10 are amino acids or peptides they comprise at least one amino acid not generally found in natural organisms.
In another aspect of this embodiment, R1 and R2 are saturated or unsaturated C10-C18 alkyl groups. In some of these compounds, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In other compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In additional compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a cytofectin formulation comprising the compounds having the above formula in a physiologically or isotonically acceptable solution.
Another embodiment of the present invention is a cytofectin formulation comprising the cationic lipids having the above formula and a co-lipid selected from the group consisting of neutral lipids, phospholipids, and cholesterol in a suitable carrier solution.
Another embodiment of the present invention is a compound having the structure: 
wherein
R1 and R2 are independently H, linear or branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4;
R3 is a linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl group having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4;
X1 and X2 are independently selected from the group consisting of NR4R5 and OR4, wherein R4 and R5 are selected from the group consisting of R1 as defined above, amino acids, peptides, polypeptides, proteins, nucleic acids, nucleotides, polynucleotides, monosaccharides, disaccharides, polysaccharide, other bioactive agents and other pharmaceutical agents;
n is 1 to 8;
m is 1 to 8;
wherein R1 and R2 are not both H.
In some aspects of this embodiment, R1 and R2 are saturated or unsaturated C10-C18 alkyl groups. In some of these compounds, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In other compounds, R3is selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein and n and m are 1-5. In further compounds, R3 is a methyl group. In additional compounds X1 and X2 are NR4R5 and R4 and R5 are H. In other compounds, n and m are 2-5.
Another embodiment of the present invention is a compound of the formula: 
wherein
R1 and R2 are saturated or unsaturated C10-C18 alkyl groups;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
R5 has the structure 
R7 and R8 are independently selected from the group defined for R1, R2, R3 and R4 and optionally further comprise a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono-, di- or polysaccharide, or other bioactive or pharmaceutical agent;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In some of these compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In other compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a compound of the formula: 
wherein
R1 and R2 are saturated or unsaturated C10-C18 alkyl groups;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
R5 has the structure 
Z is C;
R7, R8 and R9 are independently selected from the group defined for R1, R2, R3 and R4 and optionally further comprise a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono-di- or polysaccharide, or other bioactive or pharmaceutical agent;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In some of these compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In other compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a compound of the formula 
wherein
R1 and R2 are saturated or unsaturated C10-C18 alkyl groups;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
wherein R5 has the structure 
wherein
R6 or R6 together with R7 are selected from the group defined for R1, R2, R3 and R4 and optionally further comprises a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono, di- or polysaccharide, or other bioactive or pharmaceutical agent;
W is O, NR8, NH, S, or Se;
R8 is an alkyl group as defined for R1 and R2;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In some of these compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In additional compounds, R3 and R4 are methyl groups
Another embodiment of the present invention is a compound of the formula 
wherein
R1 and R2 are saturated or unsaturated C10-C18 alkyl groups;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
wherein R5 has the structure 
wherein
R6, or R6 together with R7, is selected from the group defined for R1, R2, R3 and R4 and optionally further comprises a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono, di- or polysaccharide, or other bioactive or pharmaceutical agent;
W is O, NR8, NH, S, or Se;
R8 is an alkyl group as defined for R1 and R2;
n is 1 to 6;
m is 1 to 10; and
Y is a pharmaceutically acceptable anion.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In some of these compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In additional compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a compound of the formula 
wherein
R1 and R2 are saturated or unsaturated C10-C18 alkyl groups;
R3 and R4 are independently linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0-4;
wherein R5 is NHxe2x80x94R6-R7, wherein R6, or R6 together with R7, is selected from the group defined for R1, R2, R3 and R4 and optionally further comprises a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono, di- or polysaccharide, or other bioactive or pharmaceutical agent;
n is 1-6;
m is 1-10; and
Y is a pharmaceutically acceptable anion; and
wherein if n is 1, and m is 2 to 6, and R1 and R2 separately or together are C1-C23 alkyl or C(O)xe2x80x94C1-C23, and R3 and R4 separately or together are H or unbranched alkyl C1-C6, and R5 is NHxe2x80x94R6-R7 then R6-R7 is not xe2x80x94(CH2)zNH2 where z is 2-6; or xe2x80x94(CH2)3xe2x80x94NHxe2x80x94(CH2)4xe2x80x94NH2; or xe2x80x94NHxe2x80x94(CH2)3xe2x80x94NHxe2x80x94(CH2)4xe2x80x94NH(CH2)3xe2x80x94NH2, C(O)-fluorescein, or 
where
p is 2-5, Z is H or other groups attached by amide or alkyl amino groups.
In some aspects of this embodiment, m is 2-10.
In one aspect of this embodiment, R1 and R2 are identical and are selected from the group consisting of C14H29 and C12H25. In some of these compounds, R3 and R4 are selected from the group consisting of C1-C5 alkyl groups and C1-C5 heteroalkyl groups having one heteroatom therein. In additional compounds, R3 and R4 are methyl groups.
Another embodiment of the present invention is a method of delivering an anionic molecule into a cell comprising the steps of
(a) contacting the anionic molecule with a formulation comprising an effective amount of any of the cationic lipids of the following structure to form a complex with the lipid: 
wherein
R1 and R2 are independently H, linear, branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene, or heteroalkyl groups having from 0 to 6 sites of unsaturation, or cyclic or aryl groups, said cyclic or aryl groups containing up to five heteroatoms, wherein the substituent groups are selected from the group consisting of xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein k is 0 to 4, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms;
R3 and R4 are independently linear, branched, unsubstituted or substituted C1-23 alkyl, acyl, alkene, or heteroalkyl groups having from 0 to 6 sites of unsaturation, or cyclic or aryl groups, said cyclic or aryl groups containing up to five heteroatoms, wherein the substituent groups are selected from the group consisting of xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein k is 0 to 4, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4;
R5 is absent, H or an alkyl group as defined for R1 and R2; R5 through R10 independently or in combination are absent, or are H or alkyl groups as defined for R1 and R2 and, optionally, further comprise a chemically linked amino acid, peptide, polypeptide, protein, nucleic acid, nucleotide, polynucleotide, mono-, di- or polysaccharide, or other bioactive or pharmaceutical agent;
G is absent, O, N, S or Se;
Z is O, N, S, or Se;
A is O, N, S, Se, or C;
n is 1-6;
m is 1-10;
Y is a pharmaceutically acceptable anion;
wherein if G is N and Z is O, then A is not C;
wherein if G is O and Z is O then A is not C;
wherein if G is absent, Z is O, A is O, R6 and R7 are absent, n is 1, and m is 3, then R8 is not absent or H;
wherein R1 and R2 are not both H;
and wherein if G is NH and n is 1 and m is 2 to 6, and R1 and R2 separately or together are C1-C23 alkyl or alkenyl or C(O)xe2x80x94C1-C23 alkyl or alkenyl, and R3 and R4 separately or together are H or unbranched alkyl C1-C6, and Z is O then A is not fluorescein, or 
where
p is 2-5, Z is H or other groups attached by amide or alkyl amino groups.
The method also comprises (b) contacting a cell with the lipid complex formed in step (a);
whereby a biologically effective amount of the anionic molecules are inserted into the cell. In one aspect of this embodiment, the cells are in vitro. In another aspect of this embodiment, the cells are in vivo. For example, the cells may be in an assay selected from the group consisting of murine lung transfection, murine intraperitoneal tumor, murine intramuscular and porcine or rabbit intraarterial.
Another embodiment of the present invention is a method of delivering an anionic molecule into a cell comprising the steps of
(a) contacting the anionic molecule with a formulation comprising an effective amount of any of the cationic lipids of the following formula to form a complex with the lipid: 
wherein
R1 and R2 are independently H, linear or branched, unsubstituted or substituted C1-23 alkyl, acyl, alkylene or heteroalkyl groups having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4;
R3 is a linear or branched, unsubstituted or substituted C1-23 alkyl, alkylene or heteroalkyl group having from 0 to 6 sites of unsaturation, cyclic and aryl groups, said groups comprising from 0 to 5 heteroatoms wherein said heteroatoms are not the first atoms in said groups, wherein the substituent groups are selected from xe2x80x94Oxe2x80x94(CH2)kxe2x80x94CH3, xe2x80x94Sxe2x80x94(CH2)kxe2x80x94CH3, Xxe2x80x94(CH2)kxe2x80x94, wherein X is a halide, and xe2x80x94N((CH2)kxe2x80x94CH3)2, wherein the alkyl groups of said substituents comprise from 0 to 2 heteroatoms and k is 0 to 4;
X1 and X2 are independently selected from the group consisting of NR1R5 and OR4, wherein R4 and R5 are selected from the group consisting of R1 as defined above, amino acids, peptides, polypeptides, proteins, nucleic acids, nucleotides, polynucleotides, monosaccharides, disaccharides, polysaccharide, other bioactive agents and other pharmaceutical agents;
n is 1 to 8;
m is 1 to 8;
wherein R1 and R2 are not both H.
The method also comprises (b) contacting a cell with the lipid complex formed in step (a);
whereby a biologically effective amount of the anionic molecules are inserted into the cell.